Abstract
Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Although fortilin binds and regulates various cellular proteins, the biological role of extracellular fortilin remains unknown. Here we report that fortilin specifically interacts with TGF-β1 and prevents it from activating the TGF-β1 signaling pathway. In a standard immunoprecipitation-western blot assay, fortilin co-immunoprecipitates TGF-β1 and its isoforms. The modified ELISA assay shows that TGF-β1 remains complexed with fortilin in human serum. Both bio-layer interferometry and surface plasmon resonance (SPR) reveal that fortilin directly bind TGF-β1. The SPR analysis also reveals that fortilin and the TGF-β receptor II (TGFβRII) compete for TGF-β1. Both luciferase and secreted alkaline phosphatase reporter assays show that fortilin prevents TGF-β1 from activating Smad3 binding to Smad-binding element. Fortilin inhibits the phosphorylation of Smad3 in both quantitative western blot assays and ELISA. Finally, fortilin inhibits TGFβ-1-induced differentiation of C3H10T1/2 mesenchymal progenitor cells to smooth muscle cells. A computer-assisted virtual docking reveals that fortilin occupies the pocket of TGF-β1 that is normally occupied by TGFβRII and that TGF-β1 can bind either fortilin or TGFβRII at any given time. These data support the role of extracellular fortilin as a negative regulator of the TGF-β1 signaling pathway.
Highlights
Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces
We found that α-fortilin mAb (Fig. 1a, lane 3, row a), but not IgG (Fig. 1a, lane 2, row a), immunoprecipitated fortilin and that TGF-β1 was successfully co-immunoprecipitated in the presence of fortilin (Fig. 1a, lane 3, row b) but not in its absence (Fig. 1a, lane 2, row b), suggesting that fortilin and TGF-β1 interact with each other
The most substantial findings of this study are the specific physical interaction between fortilin and TGF-β1 and elucidation of the biological significance of this interaction, namely that fortilin negatively regulates the ability of TGF-β1 to activate its canonical pathway through TGF-β receptor II (TGFβRII)
Summary
Fortilin is a 172-amino acid multifunctional protein present in both intra- and extracellular spaces. Investigation over the 20 years revealed that fortilin is a multi-functional protein implicated in diverse biological processes, including protection against apoptosis[2,3,4], endoplasmic reticulum stress handling[5], cell cycle progression[6], reactive oxygen species detoxification[7], and Ig-E-mediated histamine release[8,9]. Fortilin exists in both the cytosol and the nucleus (cellular fortilin)[2] and circulates in the blood[10] after being secreted from the cell (circulating fortilin)[11]. We propose that extracellular fortilin is an inhibitor of TGF-β1
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